Isolation of Single Cells and Uses Thereof

Abstract

The present invention relates generally to the field of immune binding proteins and method for obtaining immune binding proteins from genomic or other sources. The present invention also relates to methods and apparati for obtaining single cells that express immune binding proteins. The single cells expressing the immune binding proteins can be obtained from a patient that has had an effective immune response to a disease state (e.g., cancer or an infectious agent). The methods and apparati of the disclosure can be used to obtain immune cells that produce immune binding proteins responsible for the effective immune response. The methods and apparati of the disclosure can also be used to obtain cells that express a polypeptide (e.g., a receptor, a secreted protein, a cytokine, or a recombinant protein) or other factor of interest.

We claim: 
     
         1 . A method for obtaining the sequence of a secreted protein, comprising the steps of:
 a. obtaining a first plurality of cells that secrete a binding protein that can bind an antigen;   b. derivatizing a substrate with a plurality of carriers bound to the antigen;   c. adding to the substrate the first plurality of cells that secrete the binding proteins;   c. providing a high-throughput screening device comprising an inverted microscope and a camera component, the substrate, a cell picker component; and a robotic arm component, wherein the high-throughput screening device is capable of isolating a cell from a heterogeneous population of cells;   d. interrogating the first plurality of cells for the secreted binding protein that binds the antigen on the plurality of carriers for an optical signal that is used as a screening criteria, wherein the optical signal is associate with the plurality of carriers bound to the antigen;   e. using the high-throughput screening device to pick a second plurality of cells that are positive for the optical signal, and placing the second plurality of cells onto individual positions in a receiver plate;   f. amplifying nucleic acids from the second plurality of cells; and   g. sequencing the nucleic acids.   
     
     
         2 . The method of  claim 1 , wherein the first plurality of cells are lymphocytes. 
     
     
         3 . The method of  claim 1 , wherein the first plurality of cells are lymphocytes from a patient who has mounted an immune response. 
     
     
         4 . The method of  claim 1 , wherein the carrier of the antigen is a bead. 
     
     
         5 . The method of  claim 1  wherein the carrier of the antigen is a cell. 
     
     
         6 . The method of  claim 1 , wherein the antigen is from an infectious agent or a virus. 
     
     
         7 . The method of  claim 1 , wherein the optical signal is a change in the spatial distribution of the plurality of antigen carriers. 
     
     
         8 . The method of  claim 1 , wherein the carrier of the antigen is a cell, and binding of the cell activates a receptor which elicits the response of a fluorescent reporter, which is the optical signal. 
     
     
         9 . The method of  claim 1 , wherein a fluorescent secondary antibody binds the binding protein secreted from the first plurality of cells, wherein the fluorescent antibody emits the optical signal under stimulation. 
     
     
         10 . The method of  claim 1 , wherein a third molecule interferes with binding of the secreted binding proteins by the antigen, and the selection criteria is a decrease in signal intensity. 
     
     
         11 . The method of  claim 1 , wherein the secreted binding protein is an antibody. 
     
     
         12 . The method of  claim 1 , wherein the secreted binding protein is a soluble TCR. 
     
     
         13 . The method of  claim 1 , wherein the secreted binding protein is a soluble MHC domain. 
     
     
         14 . The method of  claim 1 , wherein the antigen is an MHC protein. 
     
     
         15 . The method of  claim 1 , wherein the antigen is a TCR. 
     
     
         16 . The method of  claim 1 , wherein the antigen is a chimeric antigen receptor. 
     
     
         17 . The method of  claim 1 , wherein the carrier is a cell and binding of the binding protein elicits a change in morphology of the carrier, which is read optically. 
     
     
         18 . The method of  claim 1 , wherein the antigen is selected from the group consisting of a hemagglutinin, a NB protein, a neuraminidase, a SARS-CoV spike protein, a coronavirus, a herpes virus, HSV gD protein, HSV gG protein, and an influenza virus. 
     
     
         19 . The method of  claim 1 , wherein the binding protein neutralizes a virus as demonstrated by the ability of the binding protein to protect susceptible cells in vitro from infection by the virus. 
     
     
         20 . The method of  claim 1 , wherein the interrogating step includes a plurality of different antigens, wherein each different antigen carrier is labeled with a different label. 
     
     
         21 . The method of  claim 20 , wherein the carrier of the antigen has a nucleic acid with a particular sequence that can be used for identification. 
     
     
         22 . The method of  claim 20 , wherein each carrier of the antigen has a fluorophore that can be used for identification of a subset of the plurality of carriers of the antigen. 
     
     
         23 . The method of  claim 20 , wherein each carrier of the antigen has a physical geometry that can be used for identification of a subset of the plurality of carriers of the antigen. 
     
     
         24 . The method of  claim 20 , wherein the carrier of the antigen is a cancer cell. 
     
     
         25 . The method of  claim 1 , wherein the carrier has antibodies that bind the secreted binding protein.